Light intensity and the rate of photosynthesis



Light intensity and the rate of photosynthesis

Students’ Sheet

Introduction

The aim is to construct an experimental set up that allows a quantitative analysis of the rate of photosynthesis under different light intensities. You should be aware that light is needed for photosynthesis and so should easily conclude that a higher light intensity leads to faster photosynthesis. However this investigation takes this concept further allowing you to explore the principles of “limiting factors” and “compensation points”.

This experimental set up actually measures the rate of photosynthesis in comparison to the rate of respiration. Because of this it is possible to determine the light intensity at which the photosynthesising material is using the same quantity of carbon dioxide in photosynthesis as it is producing in respiration. This level of light intensity is called the light compensation point and is a critical environmental tipping point for photosynthesising organisms. Above this point the light intensity is high enough to allow for growth, below this point the organism has to use up energy stores to supply the raw material for respiration.

This practical provides the opportunity for skills development in immobilisation of organic material (in this case algae but the technique is often used for enzymes as well), the use of a colorimeter, the use of a smart phone as a lux meter, and graphical skills (including plotting graphs with lines of best fit and reading off intercept points). The data from the experiment can also be used for simple computer modelling to estimate the light compensation point.

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Safety notes

Safely locate power cables of light sources to avoid them being a trip hazard.

If you are using a light source that generates a lot of heat then follow advice from your teacher with regard to setting it up, placing items in front of it and packing it away.

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Instructions

Making the algal balls

1. Place 5cm3 3% sodium alginate solution into a clean test tube

2. Place 5 cm3 concentrated algal suspension into a second, clean test tube (the algae should have been on a sunny window sill or under a bench lamp for at least 1 hour before the practical).

3. Swirl the algal suspension and then pour it into the test tube containing the sodium alginate. Stopper the tube and shake to thoroughly mix the algae and the alginate (vigorously enough so that they mix thoroughly but not too vigorously as this may trap air bubbles in the mixture).

4. Place a 12.5cm3 fine nosed syringe (with the plunger removed) vertically in the clamp stand.

5. Pour approximately 25ml 2% calcium chloride in a 50ml beaker. Place the beaker directly underneath the syringe. Adjust the height of the syringe so that the tip is approximately 10cm above the surface of the calcium chloride solution (see figure 1).

6. Pour your algae and alginate mixture into the syringe. The mixture will drip slowly into the beaker of calcium chloride (this will take 5-10mins).

7. When all the mixture has dripped through, leave the algal balls in the beaker of calcium chloride for 5-10 minutes. They will become solid. This amount of mixture, dripped from a height of 12cm, produces about 250 algal balls.

8. Tip the algal balls into a tea strainer and rinse with distilled water.

9. Place the algal balls in a beaker of fresh distilled water until you need them for the investigation (25ml in a 50ml beaker).

Preparing the Hydrogen carbonate indicator solution

10. Place 10cm3 of the concentrated hydrogen carbonate indicator solution into a measuring cylinder and make it up to 100cm3 using distilled water.

11. Use this freshly diluted indicator solution to rinse out 12 bijou bottles and their lids.

12. Place 5cm3 of the diluted indicator into each bijou bottle. Do not put the lids on the bottles, so that the indicator has more time to equilibrate with atmospheric CO2.

Investigating the effect of light intensity on the rate of photosynthesis and identifying the light compensation point

13. On a piece of graph paper, draw out a grid on which to place your tubes as in Figure 2, right.

14. Set up your lamp and grid by laying the lamp on its side and sticking the grid to the bench so that a bijou bottle at position 0 is as close to the bulb in the bench lamp as possible.

Note; with a filament bulb, a heat sink will need to be used to reduce any heating effect and this will reduce the light intensity at position 0. No heat sink is needed with the compact fluorescent and the LED bulbs, allowing the bijou bottles to be placed much closer to the lamp, exposing the algae to much higher light intensities.

15. If you are using bench lamps fitted with compact fluorescent bulbs, switch the lamp on now so that the bulb has warmed up to its maximum light intensity (this takes about 5 minutes).

16. Draw up a table for your results that has column headings for “Distance from light source /cm (or dark or control)”, “Light intensity / lux” and “Absorbance at 550nm”.

17. Strain your algal beads into a tea strainer

18. Check that everyone has reached step 16. It is best to start the experiment running as soon as possible after the first set of algal balls are added to the indicator and this requires the laboratory ceiling lights to be switched off. Given that step 19 needs to be done with the laboratory ceiling lights on, it is recommended that all students finish step 16 before you start step 19.

19. Using a spatula, place 20 beads into 11 of the 12 bijou bottles. Do not put the lids on the bottles yet. The 12th tube will be a control tube, containing indicator only.

20. Place any unused beads into a beaker of distilled water.

21. When all the students in the class are ready, switch the bench lamp on (compact fluorescent light bulbs should have been warmed up for 5 mins prior to this point [see point 15]).

22. Turn out the lab lights and measure the light intensity of each point on the grid and record the values in your results table. Light intensity can be measured using a light sensor attached to a data logger, or using a light meter app on a smart phone (there are plenty of free apps available).

23. Place the caps on the bijou bottles and place them on the grid.

24. Place bottle 12 (the control/blank) beside the bottle of algae at position 0

25. Place bottle 11 in a dark cupboard or under the lid of a cardboard box (light intensity = 0).

26. Leave the bottles for 45 minutes (30 mins is possible), swirling the contents every 5 mins and carefully replacing the bottles on their position on the grid.

27. After 45 mins, switch the lab lights on and the bench lamp off.

28. Using a clean plastic Pasteur pipette, transfer the solution from the control/blank to a clean cuvette, place into the colorimeter and use this tube to set the Absorbance at 550nm to zero. This is the colorimeter “blank”

29. Using a clean plastic pipette for each bijou bottle, transfer the well-mixed indicator solution from above the algal balls to a clean cuvette and measure the absorbance of the indicator solution at 550nm, re-setting the colorimeter to zero between each reading with the control tube as the colorimeter blank. You will need to work quickly, as the bicarbonate indicator will begin to re-equilibrate with atmospheric CO2 within a few minutes of exposure to the atmosphere. Record your values in your results table.

30. Plot a graph of Light intensity (independent variable) against Absorbance at 550nm

31. Annotate your graph to indicate:

a. The range of light intensities that were limiting the rate of photosynthesis in the algae

b. If there is a point on the graph where any further increase in light intensity doesn’t lead to a further increase in the rate of photosynthesis (light saturation point).

c. The light intensity at which there is no difference in the change in colour from the control. This is the light compensation point, where the quantity carbon dioxide being produced in respiration is equal to the quantity being used in photosynthesis.

Questions

1. The method you used

a) What organism/species did you investigate?

b) Record the conditions of your experiment – date, room temperature, number of days between making algal balls and the experiment.

c) What are the advantages and disadvantages of immobilising the algae?

d) Were your algal balls spherical and a consistent size? If so did you have to modify anything to ensure this? If not how might this have affected your results?

e) Why did you have to thoroughly mix the algae and the sodium alginate? What effect would an uneven mix have had on your experiment?

f) What happens to the sodium alginate and algae mix when it drops into the calcium chloride? Explain how this happens.

g) Why is it necessary to rinse the algal balls with distilled water?

h) Why is the hydrogen carbonate indicator necessary?

i) Why is it useful to use a colorimeter to get a numerical value for colour in this experiment?

j) How was the colorimeter set up, what settings were used and what values were recorded? Explain.

k) What other environmental factors (as well as light intensity) might vary with distance from the light source? How could you check whether they do vary and how could you control them?

l) Why do you need a control tube that contained only indicator solution?

m) Why is it important to use a clean pipette to remove the indicator from each bijou bottle at the end of the experiment?

n) Why is it important to end the experiment by switching the light source off, the laboratory ceiling lights on and removing the box covering the lid in the dark before starting to record the final colour of the solution in each bottle?

o) Why is it important to measure the final colour in each bijou bottle relatively quickly?

2. Your findings

a) Record your results in a suitable table using the column suggestions in the instructions for this investigation.

b) Describe the shape of your line of best fit in as much detail as possible (imagine there was an exam questions to describe this graph).

c) Is there a light intensity above which there is no (or very little) increase absorbance? If so what is this value?

d) Record the light intensity at which the line of best fit crosses the x-axis.

e) Using the section of data that crosses the x-axis, draw a second, enlarged graph and add a straight line of best fit through the section of data that crosses the x-axis.

f) Record the light intensity at which this straight line of best fit crosses the x-axis.

g) Comment on the two values you have obtained for where the lines of best fit cross the x-axis.

h) Calculate the equation for the straight line of best fit you have drawn (in the form y=mx+c) and use it to calculate where this line intercepts the x-axis. How does this value compare with the value you got when reading off the intercept point from your line?

i) Input your data used to plot the graph into Excel and use it to draw a graph. See if you can get Excel to draw a trend line – there are various options for the general shape of the trend line and each is effectively a model of your data. Is there an appropriate option for your data?

j) Use a subset of your data (the same subset that you used in e) to plot another graph just of the section of data that crosses the x-axis. Add a straight trend line to this graph and get Excel to show you the equation for this line. Solve the equation of the line for y=0 to identify the point where the line intercepts the x-axis (the light compensation point). Compare your hand calculated equation and intercept point with the Excel derived one.

k) Your Excel generated straight trend line is a simple computer model of your data. What assumptions does this modelling make? What are the advantages and disadvantages of modelling data in this way?

l) If the experiment was left for much too long you might expect to see that all of the tubes contained either yellow or dark purple liquid but no bottles with an intermediate colour except for the control bottle. Why would this occur and what precaution does it suggest you should take with respect to when to finish your experiment? If this did happen what would now not be possible to work out and what conclusions would you still be able to draw?

m) If the experiment was stopped too quickly how might this affect the shape of the graph and how would this affect the confidence you have in your identification of key points in the graph (the light intensities at which the line of best fit levels off and where it crosses the x-axis)?

3. The biology of what you see

a) Write a simplified word equation and balanced symbol equation for photosynthesis.

b) Explain the purpose of photosynthesis and respiration in organisms, as well as the relationship between the two processes.

c) Explain what is happening in the solution when you get a positive absorbance reading using the colorimeter.

d) Explain what is happening in the solution when you get a negative absorbance reading using the colorimeter.

e) Explain what is happening to the rate of photosynthesis and why this is occurring as light intensity increases in the part of your graph that has a relatively steep positive correlation.

f) What is a limiting factor (or rate limiting factor)? Give 4 examples of potential limiting factors for the rate of photosynthesis.

g) What is limiting the rate of photosynthesis at the light intensities where your graph shows a steep positive correlation between light intensity and Absorbance at 550nm?

h) What is a “light saturation point”?

i) If there is a light intensity above which there is no (or very little) increase in absorbance, explain why this happens. If not, do you think that if light intensity was increased further and further above what you managed in your experiment that absorbance would continue to increase and increase? Explain your thoughts.

j) What could be limiting the rate of photosynthesis at light intensities where a graph shows no increase in absorbance with an increase in light intensity?

k) What is the name of the point at which your line of best fit crosses the x-axis, what is happening at this light intensity, and what is its significance for the photosynthesising organism?

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Figure 1: equipment for making the algal balls

Box 1: Photosynthesis, respiration and carbon dioxide

Living material (including plant, animal, fungus or bacteria) is always respiring and the algae in this experiment are no exception. We assume that the rate of respiration stays constant as light intensity varies. So it is also assumed that any difference between the colour of the hydrogen carbonate indicator in the lids at different light intensities at the end of the experiment is due to a difference in the rate of photosynthesis.

This experimental set up allows you to obtain quantitative data about the rate of photosynthesis by indirectly measuring the carbon dioxide concentration in the liquid in the lids.

The “constant” rate of respiration is always adding carbon dioxide to the solution in the lid. The rate at which photosynthesis occurs determines how quickly this carbon dioxide, and the carbon dioxide that is already present in the solution, is removed from the solution.

What is actually being investigated then, is not the true “rate of photosynthesis” but the rate of photosynthesis compared to the rate of respiration. If the rate of photosynthesis is very low, then carbon dioxide may accumulate in the solution in the lid as more is being produced in respiration than is being taken up in photosynthesis. The rate at which carbon dioxide accumulates will depend on how quickly photosynthesis is occurring in relation to the rate of respiration. If the rate of photosynthesis is high enough that more carbon dioxide is taken up than is being produced in respiration then the concentration of carbon dioxide will decrease in the solution in the lid. Again, the rate at which this decrease occurs will depend on how quickly photosynthesis is occurring in relation to the rate of respiration.

Box 2: Other factors affecting colour change

The colour change is actually a measure of pH change caused by a change in CO2 concentration rather than the CO2 concentration directly. This means that anything that changes the pH away from the equilibrium, starting, pH will cause a colour change. This starting pH is 8.4 so even distilled water with pH7 is a contaminant. This however is unavoidable as the algal balls must stay damp in order for the algae to remain alive. The relatively small volume of water added with the algal balls, compared to the total volume of indicator in the bijoux bottle means that this effect is negligible in this experiment.

Box 3: From light intensity to change in colour reading

From Box 1 we can see that the rate of photosynthesis relative to the rate of respiration determines whether carbon dioxide accumulates in or is removed from the solution in the bottle, and how quickly either one occurs.

When carbon dioxide is dissolved in water it produces an acid so the concentration of carbon dioxide in the solution determines the pH of the solution.

By using hydrogen carbonate indicator solution the pH of the solution in each bijoux bottle at the end of the experiment can be measured.

A colourimeter can be used to give a quantitative measure (numerical value) of the colour rather than a qualitative measure (naming a colour, or matching to a colour chart) by eye.

The range of colours possible for the solutions at the end of this experiment is from yellow at the lower pH (more CO2 and so lower rate of photosynthesis), through red to purple at the higher pH (less CO2 and so higher rate of photosynthesis). There is a relatively linear relationship between colour (from yellow, through red, to purple) and how much green light (550nm if using a colorimeter) the solution absorbs or transmits. The yellow at the lowest pH absorbs the least and the purple at the highest pH absorbs the most.

The table below summarises the events that occur in this experiment when starting with a red indicator solution.

|Low light intensity |High light intensity |

|Low rate of photosynthesis |High rate of photosynthesis |

|Respiration produces more carbon dioxide than is used |Photosynthesis uses more carbon dioxide than is |

|in photosynthesis |produced in respiration |

|Carbon dioxide concentration in the solution increases|Carbon dioxide concentration in the solution decreases|

|The pH of the solution decreases (becomes more acidic)|The pH of the solution increases (becomes less acidic)|

|The colour of the solution changes towards the yellow |The colour of the solution changes towards the purple |

|end of the range |end of the range |

|The solution now absorbs less green light |The solution now absorbs more green light |

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